A major step towards understanding the physiological function of agonist-stimulated calcium entry channels in salivary gland cells requires identification of their molecular components and defining their regulation. TRPC (transient receptor potential canonical) proteins have been suggested as molecular candidates for store-operated calcium entry (SOCE) channels. SOCE is ubiquitously present in all cells and regulates a variety of cellular functions including salivary gland fluid secretion and inflammation. In addition other calcium channels, including TRP channels, are involved in regulating various other cellular functions such as cell growth, development. Some channels are critical mediators of cellular dysfunction. Our long term goal is to define the components that mediate and regulate Ca2+ entry into salivary gland cells. Towards this goal, our studies determine cellular mechanisms which are involved in the activation and inactivation of SOCE and define the role of TRP channels in salivary gland function as well as dysfunction. Our previous findings suggested that TRP proteins are molecular components of SOCE (TRPC1) and volume regulated Ca2+ channels (TRPV4) in salivary gland cells. We also provided evidence using TRPC1(-/-) mouse that TRPC1 accounts for more than 90% of the SOCE in SMG acini and ducts and is required for pilocarpine-stimulated saliva flow. Further, we had reported that Orai1 and STIM1 are required for TRPC1 function and that functional Orai1 was required for TRPC1-SOCE. Thus our studies have made significant advancement in our understanding of the molecular components, their assembly, and mechanism(s) of regulation of SOCE channels in salivary gland cells. We have now further assessed the molecular mechanisms involved in regulating TRPC1. Our major findings are as follows: 1.It is now well established that store-operated Ca2+ entry (SOCE) is activated by redistribution of the calcium binding protein, STIM1, from relatively diffused localization in the endoplasmic reticulum into puncta in discrete domains near the cell periphery where it interacts with and activates SOCE channels The factors involved in precise targeting of the channels and their retention at these specific microdomains are not yet defined. We had earlier investigated the nature of the plasma membrane domains that determine the sites of STIM1 aggregation and reported that lipid rafts domains (LRD) function as centers for the assembly of signaling complexes. We have reported earlier that TRPC1 is assembled in a signaling complex with key Ca2+ signaling proteins from both the ER and plasma membrane and that intact LRD are required for activation of TRPC1-mediated SOCE. Thus, our findings demonstrate that STIM1-dependent activation of TRPC1 occurs within LRD. We now report that the cholesterol-binding LRD protein Caveolin-1 (Cav1) is a critical plasma membrane scaffold that retains TRPC1 within the regions where STIM1 puncta are localized following store depletion. This enables the interaction of TRPC1 with STIM1 that is required for the activation of TRPC1-SOCE. Silencing Cav1 in human submandibular gland cells (HSG) decreased plasma membrane retention of TRPC1, TRPC1-STIM1 clustering, and consequently reduced TRPC1-SOCE, without altering STIM1 puncta. Importantly, activation of TRPC1-SOCE was associated with an increase in TRPC1-STIM1 and a decrease in TRPC1-Cav1 clustering. Consistent with this, overexpression of Cav1 decreased TRPC1-STIM1 clustering and SOCE, both of which were recovered when STIM1 was expressed at higher levels relative to Cav1. Silencing STIM1 or expression of STIM mutants with disrupted interaction with TRPC1 (ERM-STIM1 or STIM1-KK/EE) prevented dissociation of TRPC1-Cav1 as well activation of TRPC1-SOCE. Further, conditions that promoted TRPC1-STIM1 clustering and TRPC1-SOCE elicited corresponding changes in SOCE-dependent NFkB activation and cell proliferation. Together these data demonstrate that Cav1 is a critical plasma membrane scaffold for inactive TRPC1. We suggest that activation of TRPC1-SOC by STIM1 mediates release of the channel from Cav1. These important data reveal the intricate processes that regulate store-operated calcium entry. 2. We have now reported a novel relationship between cellular volume change and store-operated calcium influx. Since changes in cell volume are intricately associated with fluid secretion in salivary gland acini, we believe our studies are potentially very important and identify novel effectors of prolonged volume stress. We have shown that when cells undergo swelling due to exposure to hypotonic conditions, there is a disruption in the architecture of the ER-plasma membrane junctional region. Since this spatial positioning is critical for functioning of SOCE, we investigated the effects of hypotonic cell swelling on SOCE. Our data demonstrate that as the cell undergoes swelling, the ER recedes from the plasma membrane. This prevents the positioning of STIM1 within the optimal distance required for its interaction with the plasma membrane channels involved in SOCE. Thus SOCE is not activated under these conditions. We further reported that the reversibility of the loss in SOCE depends on the extent of hypotonic stress. At lower levels of stress, SOCE is recovered when the stress is removed the cell volume returns to normal or even after the cell undergoes normal regulatory volume decrease. We propose that factors regulated by SOCE-dependent signaling might play a role in the survival of cell to long term hypotonic stress. 3. Vesicular trafficking is a key mechanism for controlling the surface expression of TRP channels in the plasma membrane, where they perform their function. We have previously reported that TRPC3 is dynamically trafficked to the plasma membranbe in response to stimuli that lead to PIP2 hydrolysis. TRP channels in vivo are often composed of heteromeric subunits. Experiments using total internal fluorescence reflection microscopy and biotin surface labeling show that Ca(2+) store depletion enhanced TRPV4-C1 translocation into the plasma membrane in human embryonic kidney 293 cells that were coexpressed with TRPV4 and TRPC1. The translocation required STIM1. TRPV4-C1 heteromeric channels were more favorably translocated to the plasma membrane than TRPC1 or TRPV4 homomeric channels. Similar results were obtained in native vascular endothelial cells. Thus, Ca(2+) store depletion stimulates the insertion of TRPV4-C1 heteromeric channels into the plasma membrane, resulting in an augmented Ca(2+) influx in response to flow in the human embryonic kidney cell overexpression system and native endothelial cells. Since we have previously shown that TRPV4 is required for regulatory volume decrease in salivary gland cells and is trafficked during the process, we believe these new findings are also very relevant to salivary gland function. Our studies provide novel understanding of the complex regulatory mechanisms and intricate cross talk between various calcium signaling proteins and pocesses. Thus, our studies have made significant advancement in our understanding of the molecular components and molecular mechanism(s) that are involved in regulation of store operated calcium channels as well as the interaction of the SOCE system with other physiologically relevant processes that are critically involved in salivary gland fluid secretion.